1. Field of the Invention
The present invention relates to a method for regulating the permeability or integrity of the blood brain barrier and a method for delivering a compound into the central nervous system by increasing the local permeability of brain microcapillary endothelial cells constituting the blood brain barrier.
2. Description of the Related Art
The central nervous system (CNS) has been traditionally considered an “immunologically privileged site” because of the inadequacy of immune response under normal conditions. The CNS is protected by the bones of the skull, meninges, the cerebrospinal fluid (CSF), and the blood brain barrier (BBB), a highly-selective vascular compartment which limits the flow of many biologically active molecules into the CNS. The CNS has no well defined lymphatic system or mechanism for antibody production and is isolated from the immune system in the absence of disease (Leibowitz et al, 1983). This “immunological privilege” may prevent the CNS from being damaged by excessive immune responses and may deter entry of pathogens in circulating cells. However, the CNS has been shown to be constantly under immune surveillance and is capable of terminating neurotropic infections by initiating effective antigen specific and non-specific response (Cserr et al, 1992; Fabry et al 1994; Lotan et al, 1994).
The BBB functions to regulate the constitution of the brain microenvironment essential for normal cerebral functions. The permeability of the BBB is determined by complex tight intercellular junctions between a highly-specialized group of microvascular endothelial cells located within the brain which restrict passage of macromolecules between the blood and the brain (Brightman et al, 1969). This highly-selective group of microvascular endothelial cells are characterized not only by extremely tight junctions between cells, but are also surrounded by the end-feet of astrocytes, and, more rarely, by perivascular pericytes. This capillary endothelial bed is distinct from capillaries in the periphery which are not fenestrated and have underlying smooth muscle cells.
During many types of clinical conditions, the integrity of the BBB in vivo becomes impaired and nitric oxide (NO) has been implicated in this process (Boje, 1996; Buster et al, 1995; Chi et al, 1994; Johnson et al, 1995; Mayhan, 1995; Thompson et al, 1992). Other mediators, such as PGE2 and small vasoactive complement products, have also been implicated. Proinflammatory cytokines, such as TNF-α and various interleukins, are also implicated in the pathogenesis of BBB breakdown (Goldblum et al, 1990; Tracey et al, 1990). Published investigations of BBB regulation have focused on endotoxic shock as a principal model and have indicated that downstream mediators of arachidonic acid, the cyclooxygenase (COX) lipoxygenase (LOX) pathways (prostaglandins and leukotrienes, respectively) are important effector molecules. These biochemical pathways are inhibited by non-steroidal anti-inflammatory drugs (NSAIDs). The laboratory of the present inventors has previously shown that vesicular stomatitus virus (VSV) infection may result in breakdown of the BBB (Bi et al, 1995a).
Perturbations of the BBB have been reported in a wide variety of CNS disorders and diseases, and the disruption of the integrity of the BBB selectivity can lead to drastic consequences to the individual. Brain vessels are normally impermeable to serum proteins due to the presence of tight junctions. Infection of brain endothelial cells may cause perturbations in BBB function, allowing toxic substances to cross into the normally inaccessible CNS. Modern understanding of brain pathophysiology has led to the provocative thought that many diseases of the CNS are associated with a failure of BBB integrity (Pardridge, 1986) Altered BBB permeability is commonly observed during ischemia, inflammation, trauma, neoplasia, hypertension, dementia and epilepsy (Buster et al, 1995; Chi et al, 1994; Mayhan, 1995; Prado et al, 1992; Shukla et al, 1995; Zhang et al, 1995). The extravasation of plasma proteins with BBB dysfunction may occur through a number of different transcellular or paracellular routes. This includes altered tight junctions, induction of fluid-phase or non-specific pinocytosis and transcytosis, formation of transendothelial channels or by disruption of the endothelial cell membrane (Durieu-Trautmann et al, 1993; Gross et al, 1991). From a therapeutic standpoint, the selectivity of the BBB serves to prevent the entry into the CNS of therapeutic drugs. For example, in the HIV infection of microglia, AZT and protease inhibitors are excluded by the BBB. Chemotherapeutic drugs are also excluded by the BBB and conventionally require administration intraventricularly, i.e., by catheter.
Viral infections of the CNS which disrupt the integrity of the BBB include viral encephalitis, such as from polio, measles, herpes, VSV, rabies, etc. Recently, data from many laboratories, using both RNA and DNA viruses in in vitro and in vivo experimental systems, have implicated a role for NO in the immune response. The data do not indicate a magic bullet for all systems but suggest that NO may inhibit an early stage in viral replication and thus prevent viral spread, promoting viral clearance and recovery of the host.
The earliest host responses to viral infections are non-specific and involve the induction of cytokines, among them interferons (IFNs) and tumor necrosis factor alpha (TNF-α). Gamma IFN (IFN-γ) and TNF-α have both been shown to be active in many cell types and induce cascades of downstream mediators (reviewed in Levy, 1997; O'Shea, 1997; Staeheli, 1990). Others have found that NO synthase type 2 (NOS-2, iNOS) is an IFN-γ-inducible protein in macrophages, requiring IRF-1 as a transcription factor (Ding et al, 1988; Kamijo et al, 1994). The laboratory of the present inventors has observed that the isoform expressed in neurons, NOS-1, and the isoform expressed in astrocytes and endothelial cells, NOS-3, are IFN—Y, TNF-α and interleukin-12 (IL-12) inducible. Thus, NOS falls into the category of IFN-inducible proteins activated during innate immune responses.
NO, which is the smallest, lightest molecule known to act as a biological messenger in mammals, was first identified as an endothelial cell relaxing factor (Furchgott et al, 1980; Palmer et al, 1987). There are three well-characterized isoforms of nitric oxide synthases (NOS). All three enzymes have binding domains for calmodulin, flavin monocludeotide, flavin adenine dinucleotide, NADPH and a heme-binding site near the N-terminus (Table 1)
TABLE 1CellularIsoformOther Name(s)ExpressionRegulationActivityNOS-1bNOS, ncNOSneurons;Ca2+ dependentshort burstsdystrophinsoluble; consti-of smallcomplex oftuitively ex-quantity NOstriated musclepressed butalso induciblewith cytokines(IFN-γ, IL-12and TNF-α)NOS-2iNOSmacrophages;Ca2+ indepen-long burstsEBV-trans-dent; soluble;of largeformed B cells;inducible withquantity NOHeLa cellslipopoly-saccharide,IFN-γ andTNF-αNOS-3eNOS, ecNOSendothelialCa2+ de-short burstscells; astro-pendent; mem-of smallcytes,brane bound;quantity NOependymalconstituitivelycellsexpressed butalso induciblewith cytokines;estrogenresponseelement
NO has an unpaired electron; thus, its effects are mediated through other molecules that accept or share this odd electron (Butler et al, 1995; Gaston et al, 1994). Target molecules include oxygen, other free radicals, thiol groups and metals. However, NO is relatively less reactive than other oxygen radicals, such as superoxide anion (O2−) and hydroxyl radical (OH−), making it a more stable carrier of unpaired electrons.
No has a short half-life, in the range of a few seconds or less, and reacts readily with reduced cysteine moieties, yielding S-nitrosothiols that are somewhat stable with a half-life of minutes to hours. The amino acid L-arginine, a substrate for NO synthesis, contains two guanidine nitrogens that accept five electrons in an oxidation-reduction pathway, which results in the formation of L-citrulline and NO (Yun et al, 1996) (FIG. 1).
No is produced by the enzymatic modification of L-arginine to L-citrulline and requires many cofactors, including tetrahydrobiopterine, calmodulin, NADPH and O2. NO rapidly reacts with proteins or with H2O2 to form ONOO−, peroxynitrite, which is highly toxic (FIG. 1). NO also readily binds heme proteins, including Hb and its own enzyme.
The combination of NO with O2− forms peroxynitrite (ONOO−), which has the capacity to injure target cells (Beckman et al, 1996). When NO interacts with prosthetic iron groups or thiol groups on proteins, it can form complexes that activate or inactivate target enzymes. Although the action of NO is mostly local, NO has the capacity to move rapidly to distant target molecules. Unlike many messenger molecules and secretory molecules that use membrane receptors or specific supporters, NO is so lipophilic that it readily diffuses across membranes. Thus, NO can rapidly move from cell to cell, has a short range and duration of action, but exhibits high biological activity.
The neuronal NOS isoform (ncNOS, bNOS, NOS-1) is constitutively expressed and postranscriptionally regulated. Activity is dependent on calcium and calmodulin. It exists as a cytosolic homodimer under native conditions (Marletta, 1994). Enzyme levels are cytokine inducible (Barna et al, 1996; Komatsu et al, 1996). The macrophage form (NOS-2, iNOS) is rapidly induced by lipopolysaccharide (LPS), TNF-α, IL-12 and IFN-γ treatment, and is independent of calcium. NOS-2 is a cytosolic dimer under native conditions (Marletta, 1994). In the CNS, it is expressed in some astrocytes, microglia and inflammatory monocytes (Amin et al, 1995a; Galea et al, 1994; Merill et al, 1993; Zielasek et al, 1992). The endothelial form (NOS-3 ecNOS) is constitutively expressed by posttranslationally regulated and PI linked membrane associated. Like NOS-1, it is dependent on calcium and calmodulin. It is expressed in a subset of neurons and endothelial cells (Dawson et al, 1994); the laboratory of the present inventors has shown that astrocytes in the CNS synthesize NOS-3 (Barna et al, 1996) and ependymal cells (unpublished results).
Immunologically, NOS activity, NOS-immunoreactive proteins and mRNA have been found in autoimmune diseases, such as multiple sclerosis, associated with demyelinating lesions (DeGroot et al, 1997) and arthritic joints (Shiraishi et al, 1997) and are thought to contribute to disease pathogenesis. NOS is frequently observed to be induced during the immune response (Barna et al, 1996). In contrast, in many intracellular bacterial and parasitic infectious diseases, NOS activity has been observed to be essential in eliminating pathogens, such as Plasmodium falciparum (Anstey et al, 1996).
NO has been demonstrated to be a key component in host defense against a variety of pathogens, including protozoan parasites, fungi, bacteria and viruses (Harris et al, 1995; Karupiah et al, 1993; Lee et al, 1994; Seguin et al, 1994; Stenger et al, 1994; and reviewed by Reiss et al, 1998). It has inhibitory effects on ectromelia, vaccinia and herpes simplex type-1 viruses in macrophages (Karupiah et al, 1993) and the murine Friend leukemia virus (Akarid et al, 1995). It also has an inhibitory effect on HIV replication (Mannick et al, 1996).
A number of recent publications relate to the relationship between NO or NOS and the disruption or change in permeability of the BBB (Janigro et al, 1994; Dirnagle, 1996; Mayhan et al, 1996; Mayhan, 1996; Hurst et al, 1996; Chi et al, 1994; Boje 1995 and 1996; Shukla et al, 1996; Nakano et al, 1996). Boje (1995) disclosed that LPS injected into ventricles induced meningeal NO production and BBB permeability. However, the administration of amino guanidine, an inhibitor of NOS, blocked meningeal NO production and attenuated the increased permeability of the BBB observed in a rat model of meningitis. Shukla et al (1996) concluded from their results that NO itself causes an increase in the permeability of the BBB. In a study to determine whether NO mediates the selective increase in brain tumor microvessel permeability after intracarotid infusion of the vasodilator bradykinin in the RG2 rat glioma model, Nakano et al (1996) reported that transport of a tracer into brain tumors was selectively increased by the intracarotid infusion of bradykinin. Transport into normal brain was not increased. This was significantly inhibited by a NOS inhibitor, NG-nitro-L-arginine methylester. Nakano et al indicate that the selective permeability increase in brain tumor microvessels after bradykinin infusion is mediated by NO and speculate that the absence of high levels of NOS in normal brain may account for the attenuated permeability response to bradykinin in normal brain microvessels. However, the results reported in these publications on altered BBB permeability were all obtained in disease models in which the effector molecules for BBB permeability were present systemically in the animal model. There is, furthermore, no disclosure or suggestion of delivering a therapeutic compound into the CNS through increased BBB permeability by activating NOS-3.
Currently, the art has focused on regulation of NOS-3 in the periphery to control blood pressure or to relax coronary arteries during angina, and more recently, to enhance male sexual performance through sustaining erections. BBB effects are inadvertently related to gram-negative bacterial infections resulting in “shock”.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.